Method for producing microbial transglutaminase by use of pro-transglutaminase

Abstract

The invention discloses a method for producing microbial transglutaminase by use of pro-transglutaminase. The method comprises the following steps of: (1) performing gene amplification of streptoverticillium mobaraense pro-transglutaminase, and introducing the recognition sequence DDDDK of bovine enterokinase between pro-transglutaminase and mature peptide; (2) performing gene cloning to establish an expression vector, and transforming escherichia coli E.coliBL21(DE3) into engineering bacteria E.coliBL21 / proMTG, wherein the collection number of the strain is CCTCCM208240; (3) performing soluble inducible expression of pro-transglutaminase; (4) performing affinity chromatographic purification of protein to obtain pro-transglutaminase; and (5) activating the pro-transglutaminase into mature transglutaminase by use of bovine enterokinase. Through the method disclosed by the invention, the obtained transglutaminase has high bioactivity, the fermentation production technology is simplified, the sequence of pro-transglutaminase can be completely resected specifically, and the degradation of enzyme is not caused by non-specific cutting or long-time cutting.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and relates to transglutaminase genetic engineering, in particular to a method for producing microbial transglutaminase by using a transglutaminase zymogen. Background technique [0002] Microbial transglutaminase (abbreviation: MTG) is a transferase that catalyzes the transfer of acyl groups in polypeptide chains to cause covalent cross-linking reactions between proteins. It catalyzes the cross-linking reaction between protein molecules, improves the water solubility, water holding capacity, plasticity and thermal stability of protein, thereby improving the application value of protein. The enzyme has been widely used in the pharmaceutical industry, food industry and textile industry, and is considered to be the most important enzyme used in the production of new protein foods, showing significant application prospects and advantages. [0003] At present, the source of transglutami...

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Problems solved by technology

[0004] At present, the following methods are mainly used to increase the production of MTG: (1) Screening for high-efficiency enzyme-producing strains through mutation breeding, including traditional mutation breeding and space Mutation breeding, such as Japanese Ajinomoto Company [Ando H, Adachi M, Umeda K, et al. Purification and characteristics of a novel transglutaminase derived from microorganisms.Agric Biol Chem.1989,53:2613-2617] is exactly the use of induced The modified Streptomyces mohara fermented to produce MTG, but the high-yielding bacteria obtained by this method were easily degraded and the production was unstable; Wang Zhang et al. Research on Breeding onboard Shenzhou IV Spaceship. Food and Fermentation Industry, 2003, 29 (1): 6-12] Carrying MTG-producing bacteria on the "Shenzhou IV" spacecraft for breeding research, but the effect is not satisfactory; (2) Construction of engineering strains with high-efficiency expression of MTG by genetic engineering, such as Xu Bin et al. Engineering Journal. 2005, 21 (5): 794-798] Constructed genetically engineered bacteria to successfully express MTG, but the expressed MTG exists in inclusion bodies, which needs to go through processes such as washing, denaturation and renaturation, and use strong ion Purification by exchange chromatography, just obtained pure MTG, the process is loaded down with trivial details, it is difficult to realize industrialized production; Christian K etc. [Christian K. Marx, Thomas C. 2008.Hertel. expression in Escherichia coli and partial characterization of the active enzyme. Enzyme and Microbial Technology.2008,42:568-575] reported a method for soluble production of MTG, and introduced several methods that can be used to cut the transglutaminase zymogen ( Abbreviation: proMTG) enzymes, but did not change the sequence of proMTG, but directly screen the enzymes that can activate MTG, these enzymes are not specific except dispase, although dispase has strong specificity, but its efficiency not high, and The MTG after activation also has 4 amino acid residues of the zymogen at the N-terminal; Yang Huilin et al [Yang Huilin, Pan Li, Lin Ying. in Escherichia coli. Biosci Biotechnol Biochem.2009,73(11),2531-2534], Chinese patent 200810220160.1 discloses a method for Escherichia coli and its soluble expression of the transglutaminase zymogen, first carrying out high-density fermentation, and MTG is produced by activation on a purification column, but the activator used is trypsin, which is not specific, and will continue to cut proMTG after the zymogen sequence is excised, resulting in the degradation of MTG

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